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Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 926–936

Fingerprint analysis of the fruits of Cnidium monnieri extract byhigh-performance liquid chromatography–diode array

detection–electrospray ionization tandem mass spectrometry

Yi Chen a,b, Guorong Fan a,b,∗, Qiaoyan Zhang c, Huiling Wu a,b, Yutian Wu a,b

a Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, PR Chinab Shanghai Key Laboratory for Pharmaceutical Metabolite Research, No. 325 Guohe Road, Shanghai 200433, PR China

c Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, PR China

Received 4 April 2006; received in revised form 11 July 2006; accepted 7 September 2006Available online 13 October 2006

bstract

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization (ESI) and tandem mass spectrometryMS), with parallel analysis by HPLC with UV detection using a diode-array detector (DAD) was developed for the qualitative characterizationf coumarin and chromone constituents in the fruits of Cnidium monnieri. The chromatographic separations were performed on a DiamonsilTM

18 column (4.6 mm × 200 mm, 5 �m) with water with 50 mM ammonium acetate and 2% acetic acid (A) and acetonitrile (B) as the mobilehase. According to the characteristic UV spectra, the information of molecular weight and structure provided by ESI–MS/MS, 13 coumarin andchromone components were detected and identified. This method is rapid and reliable for identification of the constituents in the complex herbal

ystem, and the fragmentation patterns proposed could be extended to the similar compounds.2006 Elsevier B.V. All rights reserved.

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eywords: HPLC–DAD–ESI–MS/MS; Coumarin; The fruits of Cnidium monn

. Introduction

Coumarins containing fused benzene and �-pyrone ringsomprise a very large class of phenolic substances in plants [1].ecause of their wide applications in biology, their therapeuticctivity (bacteriostatic, anticoagulant, spasmolytic, dermatolog-cal, and mutagenic), and their use in the identification of nucleiccids and in the food and hygiene industries, these compoundsttract much interest [2–4]. Coumarins occur in over 100 plantamilies, for example, the Clusiaceae, Rutaceae, and Apiaceae.nidii fructus (she chuang zi), one of the widely used tradi-

ional Chinese medicines (TCM), is derived from mature andry fruits of the Apiaceae plant Cnidium monnieri (L.) Cus-on and has been used for treatment of pain in female genitalia,mpotence and suppurative dermatitis and is soaked in rice wines a tonic agent in China [5]. The fruit contains coumarins,

∗ Corresponding author at: Second Military Medical University, School ofharmacy, 325 Guohe Road, Shanghai 200433, PR China.el.: +86 21 2507 0388; fax: +86 21 2507 0388.

E-mail address: [email protected] (G. Fan).

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731-7085/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2006.09.015

ingerprint analysis

hromones, essential oil, terpenoids and their glycosides. Aumber of coumarins, such as xanthotoxin, isopimpinellin,ergapten, imperatorin, osthole are present in this crude drug andegarded as active principle constituents (see Fig. 1.). Xantho-oxin is used orally or topically in combination with controlledxposure to long wavelength ultraviolet radiation (UVA) or sun-ight to repigment vitiliginous skin in patients with idiopathicitiligo [6]. Previous studies have shown that naturally occur-ing coumarins (e.g., imperatorin and isopimpinellin) inhibited450-mediated enzyme activities in vitro [7]. Imperatorin and

sopimpinellin also have the potential chemopreventive effectshen administered in the diet [8]. The stimulation of melano-enesis by bergapten is related to increased tyrosinase synthesis.n addition, bergapten stimulated TRP-1 synthesis and induced aose-dependent decrease of DCT activity without modificationf protein expression [9]. Osthole could prevent postmenopausalsteoporosis [10]. It can also delay aging, build up strength,
nhance immune function, and adjust sex hormone level. Thethanol extract of the fruits of C. monnieri and the five separatend recrystallized coumarins all have growth-inhibitory effectsn the tumor cells [11].
mailto:[email protected]

dx.doi.org/10.1016/j.jpba.2006.09.015

Y. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 926–936 927

Fig. 1. Structures of the constituents identified from the fruits of C. monnieri. Chromones: (a) diosmetin (MW 300); (b) cnidimol B (MW 292); (c) dl-umtatin(MW 274); (d) cnidimol A (MW 292); (e) cnidimol E (MW 292); (f) cnidimol D (MW 292); and (g) cnidimol C (MW 208). Coumarins: (h) xanthotoxin (MW216); (i) demethyl-auraptenol (MW 246); (j) auraptenol (MW 260); (k) isopimpinelline (MW 246); (l) bergapten (MW 216); (m) 5-formylxanthotoxol (MW 230);(n) 2′-hydrate-deoxymeranin (MW 262); (o) cnidiadin (MW 314); (p) oroselone (MW 226); (q) imperatorin (MW 270); (r) osthole (MW 244); (s) cniforin A (MW375); and (t) edultin (MW 386). The structures of h, k, l, q, r were unambiguous, the others were tentatively identified.

928 Y. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 926–936

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Although the fruits of C. monnieri itself and the preparationsontaining its whole extracts were proved to be highly effec-ive for the treatment of some diseases by the pharmacologicaltudies and the clinical studies in China, they have not been offi-ially recognized internationally. One important reason for thiss the lack of an effective method to evaluate the quality of theaw medicinal materials. Establishment of the rapid and reliablenalytical methods for multi-constituents in traditional Chineseedicines catches is increasingly in the focus of researchers’

ttention.A variety of methods including high-performance liquid

hromatography (HPLC) [12], gas chromatography (GC) [13],nd micellar electrokinetic capillary chromatography (MEKC)14] have been used for characterization of the coumarins inhe fruits of C. monnieri. However, these separations only dealtith the five main coumarin components in the crude drug. It

s obvious that they are not sufficient for authenticating thiserb since perhaps all coumarin compounds are pharmacologi-ally active components in the fruits of C. monnieri. Therefore,
hemical characterization of the fruits of C. monnieri by usingnly the five main coumarins as the marker compounds seemso be insufficient, and not suitable for distinguishing the fruitsf C. monnieri from its related Apiaceae herbs. In light of
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inued ).

his, the characteristic fingerprint analysis is developed for thisurpose.

Liquid chromatography–electrospray ionization tandemass spectrometry (LC–ESI–MS/MS) is a significant analytical

ool for the identification of the known compounds and helpinghe elucidation of the unknown compounds. ESI is generallysed for molecular weight determination, and CID is performedo enhance fragmentation of the selected precursor ions. Basedn fragmentation patterns, some unknown compounds or iso-ers in complex systems could be identified [15–25].Recent success with the use of LC–ESI–MS/MS for char-

cterizing complex plant extracts suggests that the techniqueight also be effective in comprehensive characterization of

he multiple coumarin constituents in the complex herbal sys-em. The report presented here details the establishment of anC–DAD–ESI–MS/MS-based method that is capable of char-cterization of 13 coumarins and 7 chromones in one chro-atographic run in the crude extract of the fruits of C. mon-

ieri with the separation achieved both chromatographically and
ass spectroscopically. This LC–DAD–ESI–MS/MS method
nabled the simultaneous identification of the major bioactiveonstituents present in the fruits of C. monnieri, and can formhe basis for the successful quality control of this medicinal herb.

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Y. Chen et al. / Journal of Pharmaceutica

. Experimental

.1. Materials and reagents

HPLC grade acetonitrile from Merck (Darmstadt, Germany)as used. Analytical grade methanol, ethanol, acetic acid,

nd ammonium acetate were purchased from China MedicineGroup) Shanghai Chemical Reagent Corporation (Shanghai,hina). Water for HPLC analysis was purified by a Milli-Q aca-emic water purification system (Millipore, USA). Referenceompounds, xanthotoxin, isopimpinellin, bergapten, impera-orin, osthole were purchased from the National Institute forhe Control of Pharmaceutical and Biological Products (Bei-ing, China). The purity of these compounds was determined toe higher than 98% by normalization of the peak areas detectedy HPLC. Their methanolic solutions were found to be verytable. C. monnieri fruits were collected from Xinyi of Jiangsu,nd identified by Dr. Zhang.

.2. Sample preparation

The fruits of C. monnieri were ground into powder, dried at5 ◦C for 6 h. 1.0 g of the powder was ultrasonically extracted

23fa

ig. 2. Chromatograms of the fruits of C. monnieri with: (a) HPLC (320 nm); (b) Mhe peaks in chromatogram were identified by LC–DAD–MS/MS as follows: (1)

6) cnidimol D; (7) cnidimol C; (8) xanthotoxin; (9) demethyl-auraptenol; (10) aur′-hydrate-deoxymeranin; (15) cnidiadin; (16) oroselone; (17) imperatorin; (18) osth

Biomedical Analysis 43 (2007) 926–936 929

ith 20 ml 95% ethanol in a conical flask for 1 h and thenoaked at room temperature for 24 h. After filtering through

0.45 �m membrane filter, the extract was transferred into100 ml volumetric flask, and adjusted to volume with 95%

thanol.

.3. LC–DAD–ESI–MS/MS system

High-performance liquid chromatographic analysis was car-ied out using a Varian HPLC system (Palo Alto, CA, USA)quipped with a ProStar 410 autosampler, two ProStar 210umps with on-line degasser, column oven and a 335 dioderray detector (DAD). The chromatographic conditions were:iamonsilTM C18 column (4.6 mm × 200 mm, 5 �m, Dikmaechnologies, Beijing, China); sample injection volume, 20 �l;

he temperature of column oven, 30 ◦C; flow rate, 1.0 ml/min;obile phases, water with 50 mM ammonium acetate and 2%

cetic acid (A) and acetonitrile (B). A gradient programmeras used according to the following profile: 0–15 min, 35% B;
5–30 min linear increase to 80% B; 30–37 min hold on 80% B;7–40 min linear decrease to 35% B. UV spectra were recordedrom 210 nm to 500 nm, and the monitor wavelengths were sett 270 nm and 320 nm.
S–TIC (positive ion mode); and (c) MS–TIC (negative ion mode) detection.diosmetin; (2) cnidimol B; (3) dl-umtatin; (4) cnidimol A; (5) cnidimol E;

aptenol; (11) isopimpinelline; (12) bergapten; (13) 5-formylxanthotoxol; (14)ole; (19) cniforin A; and (20) edultin.


Fig. 3. MS (a) and MS/MS (b) spectra and the proposed fragmentation pattern (c) of osthole.

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A 1200L electrospray tandem mass spectrometer (Palo Alto,A, USA) equipped with an ESI interface was used for carryingut the MS and MS/MS analyses. Data were acquired and pro-essed by Varian 1200L workstation software. The electrosprayapillary potential was set to 35 V. Nitrogen was used as a dry-ng gas for solvent evaporation. The API housing and drying gasemperatures were kept at 50 ◦C and 380 ◦C, respectively. Fullcan data acquisition was performed from m/z 150 to 600 in bothositive and negative MS scan mode. MS/MS experiments wereerformed by the collision of the precursor ions with helium gas.he collision energy values were automatically selected.

. Results and discussion

.1. Selection of extraction methods

The extraction efficiency of different methods includingefluxing for 3 h, either in Soxhlet extractor or not, ultrasoni-

ppag

Fig. 4. MS (a) and MS/MS (b) spectra and the propos


ation for 1 h, and soaking for 24 h at room temperature werenvestigated. Ultrasonication and soaking were found to be suit-ble for extracting the coumarin components completely. Thextraction efficiency of various concentration of ethanol from0% to 95% was also investigated. Most of the coumarin com-onents in the herb can be extracted by 95% ethanol. On theasis of the above considerations, a two-step extraction proce-ure, ultrasonication with 95% ethanol for 1 h and then soakingor 24 h, was designed as the extraction method for the samplereparation of LC–MS/MS fingerprinting.

.2. Optimization of HPLC conditions

Ammonium acetate and acetic acid were added to mobile
hase A, to depress the tailing of the peaks of phenolic com-ounds. Concentrations of 50 mM ammonium acetate and 2%cetic acid were selected to ensure the reproducibility of the fin-erprint chromatogram. Since isocratic elution was not suitable
ed fragmentation pattern (c) of isopimpinelline.


Fig. 4. (Continued ).

Table 1HPLC–DAD–ESI–MS data and identification of constituents from the fruits of C. monnieri

Peak tR (min) λmax (nm) Ion mode Molecular ion and main fragments Identification

1 5.075 226, 255, 321 Positive 301, 324, 339, 152, 132, 124 Diosmetin2 5.379 212, 227, 319 Positive 293, 217, 253 Cnidimol B3 6.400 212, 225, 248, 264, 305 Positive 275, 297, 313, 233, 203 dl-umtatin4 10.232 210, 221, 232, 253, 295 Negative 291, 189 Cnidimol A5 10.936 214, 228, 279, 319 Positive 293, 315, 276, 222 Cnidimol E6 11.909 219, 227, 316 Positive 293, 315, 276, 262, 260, 222 Cnidimol D7 12.507 213, 220, 256, 321 Positive 209, 153 Cnidimol C8 15.296 225, 247, 299 Positive 217, 239, 255, 202, 174, 131 Xanthotoxin9 16.995 213, 221, 229, 280, 322 Positive 247, 177, 149 Demethyl-auraptenol

10 17.883 221, 321 Positive 261, 191, 163, 133 Auraptenol11 20.192 227, 266, 311 Positive 247, 269, 285, 232, 217, 189, 143 Isopimpinelline12 20.518 221, 250, 258, 310 Positive 217, 202, 188, 174, 159, 132 Bergapten13 22.104 218, 230, 322 Negative 229, 172 5-Formylxanthotoxol14 22.995 228, 289, 338 Negative 261, 245, 217, 186 2′-Hydrate-deoxymeranin15 27.645 220, 298 Negative 313, 349, 373, 243 Cnidiadin16 28.013 213, 277 Positive 227, 199 Oroselone17 28.461 217, 247, 298 Positive 271, 293, 309, 563, 203, 69 Imperatorin18 29.885 217, 255, 317 Positive 245, 267, 189, 161, 131 Osthole19 33.939 237, 256, 291 Negative 374, 433, 331, 303, 243 Cniforin A20 35.731 248, 297 Negative 445, 343, 303, 243 Edultin

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or the complete separation of coumarins in the fruits of C. mon-ieri, two sections of slopes were built into the gradient so that itovered the parts of the chromatogram where increased resolu-ion is desired. This gradient of the mobile phase could achieve

aximum throughput and optimal resolution. The wavelengthor the detection of the components in the fruits of C. monnierias selected by using a DAD detector. It was found that the

hromatograms at 270 nm and 320 nm could well represent therofile of the constituents. The representative chromatogram at20 nm is shown in Fig. 2a. The typical on-line UV–vis spectraf four respective coumarins, xanthotoxin, bergapten, impera-orin, and osthole, recorded using a DAD detector is also shown.

.3. HPLC–MS/MS analysis

The MS spectra were detected in both the positive and nega-ive ion mode and their TIC chromatograms are shown in Fig. 2b

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Fig. 5. MS (a) and MS/MS (b) spectra and the pro


nd c, respectively. In MS spectra, most of the constituents exhib-ted their quasi-molecular ions [M + H]+, adduct ions [M + Na]+

nd [M + K]+ in positive ion mode, while exhibited [M−H]−,M + Cl]−, and [M + CH3COO]− in negative ion mode. The pos-tive ion mode was found to be more sensitive and of loweroise to the most coumarins in the fruits of C. monnieri. Basedn the m/z value, UV spectra and the comparison with stan-ard compounds, five peaks were unambiguously identified asanthotoxin (8), isopimpinelline (11), bergapten (12), impera-orin (17), and osthole (18). Other 15 peaks were tentativelydentified as diosmetin (1), cnidimol B (2), dl-umtatin (3), cni-imol A (4), cnidimol E (5), cnidimol D (6), cnidimol C (7),emethyl-auraptenol (9), auraptenol (10), 5-formylxanthotoxol

′
13), 2 -hydrate-deoxymeranin (14), cnidiadin (15), oroselone16), cniforin A (19), and edultin (20) by comparing their m/zalue and UV spectra with the literature data [26–34]. The resultsre listed in Table 1 and the structures of these compounds are
posed fragmentation pattern (c) of edultin.


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hown in Fig. 1. It could be seen that the constituents in the fruitsf C. monnieri detected in these assay were coumarins (8–20)nd chromones (1–7), while the types of the coumarins includedimple coumarin (9, 10, 18), linear furocoumarin (8, 11–14, 17),nd angular furocoumarin (15, 16, 19, 20).

Fig. 3 shows the MS and MS/MS spectra and the proposedragmentation pattern of peak 18, a simple coumarin. The MSpectra in the positive mode exhibited an abundant parent ionM + H]+ at m/z 245 and ion [M + Na]+ at m/z 267, while a frag-ent ion at m/z 189 was also observed. By the optimum cone

oltage setting at 20 eV, the fragment ion at m/z 189 could bettributed to the loss of C4H8 from the parent ion. This was fur-her fragmented to yield an ion with m/z 161, signaling loss ofO. Subsequent loss of 30 mass units equating to the loss ofH2O occurred with a peak observed at m/z 131.

A fragmentation pathway of linear furocoumarin was pre-ented by the fragmentation pattern of peak 11. An abundantarent ion [M + H]+ at m/z 247 and ion [M + Na]+ at m/z 269ere produced. This parent ion fragmented initially with twice

oss of methyl (m/z 232 and m/z 217), followed by loss of 28ass units attributed to CO (m/z 189), and finally generated a

eak at m/z 143 by loss of 46 mass units (see Fig. 4.).For the angular furocoumarin, such as peak 20, once again

parent ion [M + CH3COO]− at m/z 445 was observed in theegative mode, which dissociated in MS/MS to generate ionst m/z 343, 303 and 243. A loss of C4H7CO and CH3CO wasbserved in no sequence (see Fig. 5.).

. Conclusion

Combining the HPLC separation, the UV absorption patternnd the information of molecular weight and structure provided

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inued ).

y ESI–MS/MS, HPLC–DAD–ESI–MS/MS was proved to ben effective tool for the study of a complex herbal system. Thisechnique showed the superiority and became a key strategy innalyzing traditional Chinese medicines.

According to the principle to choose the relevant marker com-ounds, which are specific to species, taxonomic evidences oftability, specificity, discontinuity and limitation, integrality andorrelative differences of the marker compounds were signifi-ant. The results in our study showed that thirteen coumarinsncluding simple coumarin, linear furocoumarin, and angularurocoumarin and seven chromones were coexistent in the fruitsf C. monnieri. Although some other plants, especially in Api-ceae, and Rutaceae, may have more or less coumarins andhromones as in the fruits of C. monnieri, no report was found onhe simultaneous occurrence of the three types of coumarins andhe chromones in other plants. For example, ostheol, bergapten,anthotoxin, isopimpinellin, and imperatorin were all presentn the roots of Heracleum rapula [46], while the chromonesere not found. This is a significant characteristic in the fin-erprint chromatogram of the fruits of C. monnieri. Then thether tentatively identified coumarins and chromones in the fin-erprint chromatogram can assist to identify the plant species.he compounds in other related plants are shown in Table 2

35–53].In this study, the chemical constituents in the fruits of C.

onnieri were analyzed by HPLC–DAD–ESI–MS/MS. Ana-yzed by direct infusion, the fragmentation patterns of referenceompounds were proposed. Based on the fragmentation pat-
erns and the comparison of the UV, MS data of sample withhe published literature data, twenty constituents in the fruits of. monnieri were identified. Among them, five main coumarinsere unequivocally and the others tentatively identified. The fin-

Y. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 926–936 935

Table 2Distribution of the coumarins and chromones in the representative plants of Apiaceae and Rutaceae

Plant Coumarin Chromone Reference

Simple coumarin(osthole)

Linear furocoumarin Angularfurocoumarin

Xanthotoxin Isopimpinelline Bergapten Imperatorin

Angelica dahurica√ √ √ √

[35,36]Angelica pubescence

√ √ √ √[37,38]

Angelica morri√ √

[39,40]C. monnieri

√ √ √ √ √ √ √[41]

Cnidium dahuricum√

[42]Peucedanum ostruthium

√ √[43,44]

Peucedanum acaule√ √

[45]H. rapula

√ √ √ √ √[46]

Heracleum stenopterum√ √ √

[47]Saponikvia cata

√ √ √ √[48]

Bupleurum chinese√

[49]Murraya siamensis

√[50]

Murraya exotica√

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anthoxylum dimorphophyllumanthoxylum shinifolium

erprint chromatograms can be useful to ensure the safety andfficacy, and to optimize the quality control of the fruits of C.onnieri.

cknowledgement

This work was supported by Fundamental Research Keyroject founded by Science and Technology department ofhanghai, PR China, Grant No. 03JC14005.

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